The overall objective of my project is to characterize the nature of basic mechanisms operating in the replication of DNA. My approach exploits 6(p-hydroxy-phenylazo)-uracil (HPUra), a novel, selective inhibitor of semi-conservative DNA replication in GR plus bacteria. I have found that HPUra can inhibit in vitro the ATP-dependent, semi-conservative replication of DNA catalysed by toluene-treated Bacillus subtilis. I will initially exploit toluene-treated B. subtilis (1) to study the effects of HPUra on the metabolism and function of ATP and the deoxyribonucleotide precursors of DNA, and (2) to identify and characterize HPUra-resistant mutants which are specifically altered in the drug-sensitive process or "site" required for DNA replication. Exploiting appropriate HPUra-resistant mutants and information derived from studies with toluene-treated cells, I will attempt to identify the drug-sensitive site. Initially, I will attempt to prepare a cell-free system capable of catalysing HPUra-sensitive DNA synthesis in vitro. If sucessful, I will attempt to isolate the HPUra-sensitive component and characterize its function as a cell structure, or enzyme essential to DNA replication. I will consider the function of novel DNA polymerases, DNases, and "unwinding" proteins as potential targets for HPUra binding and inhibition.